Synthetic ligands for the differentiation of closely related toxins and pathogens

ABSTRACT

Synthetic ligand compounds and methods of differentiating between Shiga toxin 1 and Shiga toxin 2 are disclosed herein. Another embodiment includes a kit for differentiating between Shiga toxin 1 and Shiga toxin 2. Assay systems and methods for providing an assay are also provided for herein.

REFERENCE TO RELATED APPLICATION

The present application claims priority of U.S. Provisional ApplicationSer. No. 60/923,651, filed Apr. 16, 2007, the entire disclosure of whichis hereby incorporated herein by reference.

TECHNICAL FIELD

Compounds and methods of differentiating between closely related toxinsand pathogens and differentiation between serotypes of the same toxin(or pathogen) family. More particularly, synthetic ligands and methodsof using such synthetic ligands to differentiate between Shiga toxin 1and Shiga toxin 2.

BACKGROUND

Multivalent ligands have been shown to capture toxins and pathogens.However, these conventional compounds and techniques have not providedthe selectivity necessary to differentiate between closely relatedtoxins or pathogens. Conventional ligands utilized for sensing toxins orpathogens have been full-length antibodies that possess very highspecificity and binding affinities. Such antibodies are not ideal asthey are not thermally, chemically and biologically stable enough tolast for long periods of time. For example, in diagnostic applicationsfor many pathogens, the constant genetic drift renders antibodiesineffective as their specificity and binding affinities decrease overtime. Moreover, the presence of antibody matrix effects from a host'simmune response can further interfere with detection in clinical samplesand again render antibody capture unreliable.

SUMMARY

In accordance with one embodiment, a compound for detecting varianttoxins and pathogens, the compound comprising the general formula (I):

wherein: n equals 1, 2, 3, 4, 5 or 6; B═O, NH, S, SO, SO₂, or P(O)R,C═NH₂, COOH, biotin or derivatives thereof, and A comprises aglycoconjugate, wherein the glycoconjugate is selected from the groupconsisting of:

wherein R comprises H, Ac or derivatives thereof; X═OH, SH, NHAc, NHCF₃,NH₂, NHCH(═NH)NH₂, or derivatives thereof; Y═OH, NHAc, SH, NHCF₃, NH₂,NHCH(═NH)NH₂ or derivatives thereof; Z═OH, NHAc, SH, NHCF₃, NH₂,NHCH(═NH)NH₂ or derivatives thereof.

In accordance with another embodiment, a glycoconjugate consistsessentially of:

wherein R comprises H, Ac or derivatives thereof; X═OH, SH, NHAc, NHCF₃,NH₂, NHCH(═NH)NH₂ or derivatives thereof; Y═OH, NHAc, SH, NHCF₃, NH₂,NHCH(═NH)NH₂ or derivatives thereof; Z═OH, NHAc, SH, NHCF₃, NH₂,NHCH(═NH)NH₂ or derivatives thereof, and wherein the glycoconjugate hasa sufficient affinity to bind to a Shiga toxin for enterotoxigenic E.coli, wherein (b) binds with Shiga toxin 1 and (a) and (c) bind withShiga toxin 2.

In accordance with yet another embodiment, a kit for detecting a toxincomprises at least one container containing at least one capture agent,wherein the at least one capture-agent substantially only binds to Shigatoxin 1 or Shiga toxin 2.

BRIEF DESCRIPTION OF THE DRAWINGS

While the specification concludes with claims particularly pointing outand distinctly claim the invention, it is believed that the same will bebetter understood from the following description taken in conjunctionwith the accompanying drawings in which:

FIG. 1 illustrates three embodiments of synthetic ligands;

FIG. 2 illustrates the synthesis of a di-N-acetylgalactosaminederivative;

FIG. 3 generally represents a compound arrangement for a high affinitymultivalent ligand;

FIG. 4 illustrates stepwise formation of a synthetic ligand;

FIG. 5 illustrates another stepwise formation of a synthetic ligand;

FIG. 6 illustrates another stepwise formation of a synthetic ligand;

FIG. 7 generally depicts an assay for detection of a toxin using asynthetic ligand;

FIG. 8 a represents a line graph illustrating the binding relationshipbetween Compound B and C with the variants of enterotoxigenic E. coli,Shiga toxin 1 or Shiga toxin 2;

FIG. 8 b represents a line, graph illustrating the binding relationshipbetween Compound A with the variants of enterotoxigenic E. coli, Shigatoxin 1 or Shiga toxin 2;

FIG. 9 a illustrates a biological assay system for detecting thepresence of a particular toxic substance;

FIG. 9 b illustrates a biological assay system for detecting thepresence of a particular toxic substance;

FIG. 10 a represents a line graph illustrating detection of Shiga toxin1 or Shiga toxin 2 in food products using one synthetic ligand;

FIG. 10 b represents a line graph illustrating detection of Shiga toxin1 or Shiga toxin 2 in food products using another synthetic ligand;

FIG. 11 a represents a line graph illustrating detection of Shiga toxin1 or Shiga toxin 2 in food products using one synthetic ligand; and

FIG. 11 b represents a line graph illustrating detection of Shiga toxin1 or Shiga toxin 2 in food products using another synthetic ligand.

DETAILED DESCRIPTION

Detection of the Shiga toxin producing E. coli, and diagnosis of diseasein clinical settings presents a challenge. Isolation of E. coli isclinically significant under special circumstances and is dependent onthe pathogenic potential of the E. coli strains, mainly because somestrains produce essentially harmless forms of E. coli while other,particularly Shiga toxins, can produce life-threatening diseases (i.e.,hemorrhagic colitis and hemolytic uremic syndrome, etc.). Thus, it isimportant to develop diagnostic tests to detect Shiga toxin todistinguish harmless E. coli from isolates that are capable of causinghuman disease. In addition to detecting the presence of Shiga toxin, itis also important to differentiate between the type of Shiga toxin thatis produced. Today, there are two major antigenic groups of Shigatoxins, Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). There are alsoseveral minor antigenic variants, Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, forexample.

The variants of the Shiga toxins can have very different potencies,particularly related to the impact such toxins have on people. Forexample, Shiga toxin 2 is more toxic than Shiga toxin 1 for primates.Thus, the development of a compound and method of distinguishing betweensuch variants (such as Stx1 and Stx2) provides a significant advantagein diagnosing and treating the effects of such toxins or pathogens.

Toxins and pathogens (including viruses and bacteria) have been known tobind to cell-surface glycolipids, however, variants of these toxins orpathogens have different binding affinities for closely relatedglycolipids. These differences in receptor recognition influence whichcells will be targeted by the toxin or pathogen and ultimately includethe outcome of the potential disease. Such toxins (including theirsaccharide specificity) include botulinum neurotoxins (gangliosidesGD1a, GD1b, GT1a), Ricin (Galactose, N-Acetylgalactose), Shiga toxin 1(Gal (α1,4) Gal(β1,4)Glc-ceramide or globotriaosylceramide (Gb3)), Shigatoxin 2 (analogues of Gb3, GalNAc (α1,4) Gal(β1,4)Glc-ceramide), Shigatoxin 2e (GalNAc(α1,3) Gal (α1,4) Gal(β1,4)Glc-ceramide (Gb4)),clostridium perfringens epsilon toxin (gangliosides GM1, GM3),staphylococcal enterotoxin B (SEB) Gal (1,4) Gal-ceramide), pertussistoxin (sialic acid, Gal(β1,4)Glc-ceramide, gangliosides), cholera toxin(Ganglioside GM1), E. coli enterotoxin, LT-I (Ganglioside GM1), E. colienterotoxin, LT-IIa (Gangliosides GD1b, GD1a, GM1), E. coli enterotoxin,LT-IIb (Ganglioside GD1a). The compounds described and claimed hereinhave been developed to have high selectivity and sensitivity which allowthem to bind to specific toxins.

Embodiments are herein described in detail in connection with thedrawings of FIGS. 1-11, wherein like numbers indicate the same orcorresponding elements throughout the drawings.

The development of synthetic ligands that mimic the natural (orunnatural) receptors associated with the variants for toxins andpathogens has provided for the capacity to differentiate between closelyrelated toxins or pathogens. Examples of some embodiments of suchsynthetic ligands can be found in FIG. 1. The compounds shown in FIG. 1include synthetic ligands which are designed to differentiate betweenclosely related toxins. For example, Compound A has been designed tobind to Shiga toxin 1, but not Shiga toxin 2, while Compounds B and Chave been designed to bind to Shiga toxin 2, but not Shiga toxin 1. Asmentioned herein, having such compounds readily capable ofdifferentiating between variant forms of toxins and pathogens allows forthe diagnosis and treatment of any diseases specifically related totheses variant forms of the toxins or pathogens.

The embodiments of the compounds illustrated in FIG. 1 each includevarious types of glycoconjugates, which have been configured to bind tovariant forms of the Shiga toxin. It will be understood that theseglycoconjugates can be produced using various process methods, however,for purposes of illustration, one embodied method for the synthesis of adi-N-acetylgalactosamine derivative (i.e., allyl(2-N-acetamido-2-deoxy-3,4,6 tri-O-acetyl α-D-galactopyranosyl)-(1→4)2-N-acetamido-2-deoxy-3,6 di-O-benzyl-β-D-galactopyranoside) isillustrated in FIG. 2 and discussed herein. The synthesis of thedi-N-acetyl galactosamine was generally achieved by coupling an acceptorobtained by a 2-step procedure fromallyl-e-deoxy-2-azido-4,6-benzylidene-β-D-galactopyranoside, withtrichloroacetimidate donor in the presence of catalytic amount of TMSOTfyielding the disaccharide form of the di-N-acetylated galactosederivative.

The synthetic ligands which are discussed herein generally have threecomponents which include a recognition element, spacer (which can beterminated in an azide) and a dimeric scaffold bearing two alkynes allof which are generally represented by the embodiment illustrated in FIG.3. The embodiment of the ligand represented in FIG. 3 has tworecognition elements. The recognition elements are adapted to bind totoxins and pathogens and can include antibodies, antibody fragments,aptamers, carbohydrates, peptides DNA or RNA. Moreover, the tworecognition elements represented in FIG. 3 can be the same or different.The spacer illustrated in FIG. 3 can vary in length and can be a factorin increasing the selectivity or; affinity the synthetic ligand has fora particular toxin or pathogen. The embodiment of the scaffold shown inFIG. 3 is a multivalent dimeric scaffold. The dimeric scaffold affordseasy access to multivalency by virtue of one tetramer binding to fourbiotins.

The following examples provide three different embodiments directed tothe synthesis of the three compounds shown in FIG. 1.

EXAMPLE 1 Synthesis of Compound C

A synthesis of Compound C is illustrated in FIG. 4. The di-N-acetylatedgalactose derivative as shown in FIG. 2 can be processed to form theembodied synthetic ligand compound (VI) illustrated in FIG. 4. Theprocess of synthesizing compound (VI) as shown in FIG. 4 includes havingthe di-N-acetylated galactose derivative (I) (i.e., Allyl(2-N-acetamido-2-deoxy-3,4,6 tri-O-acetyl α-D-galactopyranosyl)-(1→4)2-N-acetamido-2-deoxy-3,6 di-O-benzyl-β-D-galactopyranoside) modified sothat the azide functionalities were reduced to the N-acetyl groups usingthiolacetic acid.

Compound (I) (116 mg, 0.15 mmol) and(1,1-Dimethylethyl)dimethyl(4-pentenyloxy)silane (300 mg, 1.5 mmol) weredissolved in 15 ml CH₂Cl₂ andbenzylidene-bis(tricyclohexylphosphine)dichlororuthenium (Grubb's 1stgeneration catalyst, 28 mg, 0.034 mmol) was added to it under argonatmosphere. The resulting orange colored solution was refluxed for 16 h.The reaction mixture was then cooled to room temperature, the solventwas removed in vacuo and the crude product was purified by flash columnchromatography, eluting with a 80:20 mixture of EtOAc:hexane, to giveCompound (II) (i.e., 6-[(1,1-Dimethylethyl)dimethylsilyl]oxy]-2-en(2-N-acetamido-2-deoxy-3,4,6tri-O-acetyl-α-D-galactopyranosyl)-(1→4)-2-N-acetamido-2-deoxy-3,6di-O-benzyl-O-D galacto pyranoside) as a white solid (123 mg, 87%).HRMS: Calculated for [C₄₈H₇₀N₂O₁₅Si+H]⁺: 943.4619. Found 943.4666.

Compound (II) (100 mg, 0.032 mmol) was dissolved in THF (2 ml) andcooled to 0° C. A solution of TBAF in THF (0.2 ml of 1 M solution inTHF, 0.127 mmol) was added drop wise and the resulting solution wasstirred for 3 h at room temperature. The reaction was quenched usingsaturated NaHCO₃ solution and the product was extracted with 2×25 mlEtOAc. The organic layer was collected, dried over anhydrous Na₂SO₄,filtered and the solvent was removed in vacuo. The crude product waspurified by flash column chromatography, eluting with a 10:90 mixture ofMeOH and EtOAc, to give the alcohol as a white solid (76 mg, 86.9%).HRMS Calculated for [C₄₂H₅₆N₂O₁₅Na]⁺851.3578. Found 851.3595. Next, thealcohol (90 mg, 0.109 mmol) and diisopropyl ethyl amine (0.270 ml, 0.155mmol) were dissolve 4 in CH₂Cl₂ (15 ml) and cooled to −10° C. Methanesulfonyl chloride (0.1 ml, 129 mmol) was added drop wise and theresulting solution was stirred for 1 h slowly warming to roomtemperature and further stirred at room temperature for 4 h. Water wasadded to the solution and the product was extracted with 2×25 ml CH₂Cl₂.The organic layer was collected, dried over anhydrous Na₂SO₄ and thesolvent was removed in vacuo to give mesylated product which was used innext reaction without purification. The mesylated intermediate andsodium azide (100 mg, 1.53 mmol) were dissolved in 3 ml of DMF and theresulting solution was stirred at 65° C. for 5 h. The reaction mixturewas then cooled to room temperature and the product was extracted with2×25 ml EtOAc. The organic layer was collected, dried over anhydrousNa₂SO₄, filtered and the solvent was removed in vacuo and the crudeproduct was purified by flash column chromatography, eluting with 100%EtOAc, to give Compound (III) (i.e., 6-Azido-2-en(2-N-acetamido-2-deoxy-3,4,6 tri-O-acetyl-α-D-galactopyranosyl)-(1→4)-2-N-acetamido-2-deoxy-3,6-di-O-benzyl-β-D-galactopyranoside)as a white solid (62 mg, 68.9% over 3 steps). HRMS Calculated for[C₄₂H₅₅N₅O₁₄Na]⁺: 876.3643. Found: 876.3663.

Compound (III) (10 mg, 0.028 mmol), Compound (II) (54 mg, 0.063 mmol),sodium ascorbate (14 mg, 0.071 mmol), and CuSO₄ (9 mg, 0.036 mmol) weremixed in a 1:1 mixture of water and THF (3 ml) was stirred at roomtemperature for 24 h. After evaporation of the solvents, the crudeproduct was directly loaded onto a silica gel column and the product waspurified by flash column chromatography, eluting with 85:15 mixture ofEtOAc and CH₃OH (methanol), to give Compound (IV) as a white solid (50mg, 86%). HRMS Calculated for [C₁₀₃H₁₃₁N₁₃O₃₂+2H]²⁺: 1031.9584 Found:1031.9696.

Compound (IV) (45 mg, 0.022 mmol) was dissolved in CH₃OH (10 ml) andPd(OH)₂ on carbon (30 mg) was added to it. The reaction mixture wasstirred under hydrogen atmosphere under 1 atm pressure and at roomtemperature for 12 h. The catalyst was filtered through celite and thesolvent was removed under vacuo to yield the debenzylated intermediate.The tetrahydroxide was dissolved in 3 ml of dry pyridine; catalyticamount of DMAP (5 mg) was added to it and cooled to 0° C. Aceticanhydride (1.5 ml) was then added to it at 0° C. After stirringovernight, the solvent was removed in vacuo and the residue wassubjected to column chromatography, eluting with to give Compound (V) asa white solid (36 mg, 88%). HRMS Calculated for [C₈₃H₁₁₉N₁₃O₃₆+2H]²⁺:937.9013 Found: 937.9031.

Compound (V) (10 mg, 5.33 mmol) was taken in dry CH₂Cl₂ (2 ml) and TIPS(0.020 ml) was added to it via syringe. TFA (0.100 ml) was added dropwise and stirred at room temperature for 12 h. Saturated NaHCO₃ solutionwas used to quench the reaction and the compound was extracted in 2×25ml CH₂Cl₂. The organic layer was dried over anhydrous Na₂SO₄ and thesolvent was removed in vacuo. The crude product was purified by flashcolumn chromatography, eluting with a 1:4 mixture of hexane and EtOAc,to give the free amine as a white solid. This product was used withoutfurther purification in the next step. CDMT (2 mg, 0.011 mmol) wasdissolved in dry THF (0.5 ml) cooled to 0° C. and NMM (0.010 ml) wasadded to it and stirred for 30 min at 0° C. Biotin (2.2 mg, 0.009 mmol)in DMF (0.5 ml) was added dropwise to the mixture and the mixture wasreacted overnight at 0° C. under continuous stirring. The amine (8 mg,0.0045 mmol) and NMM (0.010 ml) in DMF:THF (0.5 ml, 1:1) were addeddropwise to the mixture under stirring at 0° C. The mixture was reactedfor 20 h slowly warming to room temperature. Water was added drop wiseto the mixture while stirring and the compound extracted in 2×25 ml ofEtOAc. The organic layers were dried over anhydrous Na₂SO₄, filter andsolvent removed in vacuo. The residue was purified by columnchromatography, eluting with a 1:9 mixture of methanol and EtOAc, togive Compound (VI) as a white solid (5.5 mg, 61%). HRMS Calculated for[C₈₈H₁₂₅N₁₅O₃₆S+2H]²+: 1000.9139. Found 1000.9156.

Compound (VI) (4 mg, 0.002 mmol) was dissolved in CH₃OH (2 ml) and asolution of NaOMe in CH₃OH (0.7 M, 0.5 ml) was added and the reactionmixture was stirred at room temperature for 16 h. The reaction wasneutralized by careful addition of Amberlite-15H′ resin and the resinwas filtered. The solvent was removed in vacuo and the residue waspurified by Biogel P-2 gel column chromatography, using water as eluent.The product was lyophilized to give Compound C, where R═H, as a whitesolid (2.7 mg, 86%). HRMS Calculated for [C₆₈H₁₀₅N₁₅O₂₆S+2H]²⁺:790.8612. Found 790.8580.

EXAMPLE 2 Synthesis of Compound B

A synthesis of Compound B is illustrated in FIG. 5. Compound (VIII) isformed from a trichloroacetimidate (210 mg, 0.22 mmol) and an acceptor(Compound (VII) (120 mg, 0.26 mmol) were dissolved in CH₂Cl₂ (15 ml) andcooled, to −20° C. TMSOTf (0.093 ml of a 0.22 M solution in CH₂Cl₂,0.022 mmol) was added dropwise via syringe and the resulting solutionwas stirred for 1.5 h at −20° C. Upon completion (by TLC), the reactionwas quenched using cold saturated NaHCO₃ solution and the product wasextracted with 2×25 ml CH₂Cl₂. The organic layer was collected, driedover anhydrous Na₂SO₄, and the solvent was removed in vacuo. The crudeproduct was purified by flash column chromatography, eluting with a 1:1mixture of hexane and EtOAc, to give the coupled product as a whitesolid (194 mg, 60.0%). HRMS Calculated for [C₇₃H₇₉N₃O₁₈+Na]⁺1308.5256.Found: 1308.5281. This white solid was dissolved in thioacetic acid (1.5ml) and the resulting solution was stirred for 48 h at room temperature.The solvent was removed in vacuo and the crude product was purified byflash column chromatography, eluting with 100% EtOAc, to give Compound(VIII) (i.e., Benzyl (2-N-acetamido 2-deoxy3,4,6-tri-O-acetyl-α-D-galacto pyranosyl) (1→4)(2,3,6-tri-O-benzyl-β-D-galactopyranosyl)(1→4)2,3,6-tri-O-benzyl-β-D-glucopyranoside) as a white solid (105 mg,53.5%). HRMS Calculated for [C₇₅H₈₃NO₁₉+Na]⁺1324.5457. Found: 1324.5483.

Compound (VIII) (105 mg, 0.081 mmol) was dissolved in CH₃OH (10 ml) andPd(OH)₂ on carbon (30 mg) was added to it and the reaction mixture wasstirred under 1 atm hydrogen atmosphere at room temperature for 12 h.The catalyst was filtered using celite and the solvent was removed undervacuo to yield the debenzylated product as a white solid. The solidmaterial was dissolved in 8 ml of dry pyridine, catalytic amount of DMAP(5 mg) was added to it and cooled to 0° C. Acetic anhydride (2.5 ml) wasadded to it and stirred overnight. The solvent was removed in vacuo andthe residue was subjected to column chromatography, eluting with 100%EtOAc to give Compound (IX) (i.e., Acetyl (2-N-acetamido 2-deoxy3,4,6-tri-O-acetyl-β-D-galacto pyranosyl) (1→4)(2,3,6-tri-O-acetyl-β-D-galactopyranosyl)(1-4)2,3,6-tri-O-benzyl-β-D-glucopyranoside) as a white solid (63 mg, 81.5%over 2 steps). HRMS Calculated for [C₄₀H₅₅NO₂₆+Na]⁺988.2910. Found:988.2965.

Compound (IX) 130 mg, 0.135 mmol) was dissolved in 3 ml of anhydrous THFand NH₂NH₂.HOAc (15 mg, 0.162 mmol) was added to it. The reaction wasstirred at room temperature for 6 h. The reaction mixture was dilutedwith 5 ml of EtOAc and 5 ml of water was added the organic layer wasseparated and dried in vacuo to give the hemiacetal (105 mg, 85%), whichwas directly used in the next step. Anhydrous K₂CO₃ (400 mg, 2.89 mol)was added to the solution of hemiacetal 120 mg, 0.129 mmol) andtrichloroacetonitrile (100 μL, 1.0 mmol) in CH₂Cl₂ (3 ml) at roomtemperature. The reaction mixture was stirred at room temperature for 8h, washed with water and the organic layer was dried over anhydrousNa₂SO₄, filtered and concentrated in vacuo. The residue was purified bycolumn chromatography, eluting with EtOAc, to give the trichloroimidateas a pale yellow solid (121 mg, 87%). The imidate (64 mg, 0.060 mmol)and 1-azido hexanol (17 mg, 0.12 mmol) were dissolved in CH₂Cl₂ (2 ml)and cooled to 0° C. A 0.22 M solution of TMSOTf in CH₂Cl₂ (0.055 ml,0.012 mmol, 0.2 eq.) was added drop wise and the resulting solution wasstirred for 1.5 h at 0° C. The reaction was quenched by saturated NaHCO₃solution (cold) and extracted with CH₂Cl₂. The organic layer was driedover anhydrous sodium sulfate, filtered, concentrated in vacuo andpurified by column chromatography, eluting with EtOAc to give Compound(X) (i.e., 1-Azido-hexyl(2-N-acetamido 2-deoxy3,4,6-tri-O-acetyl-α-D-galacto pyranosyl) (1→4)(2,3,6-tri-O-acetyl-β-D-galactopyranosyl) (1→4)2,3,6-tri-O-acetyl-β-D-gluco pyranoside) as a syrupy solid (28 mg, 46%).HRMS Calculated for [C₄₄H₆₄N₄O₂₅+Na]⁺: 1071.3764. Found: 1071.3786.

Compound (X) (14 mg, 0.0137 mmol), a biotin (3 mg, 0.0062 mmol), sodiumascorbate, (3 mg, 0.015 mmol), and CuSO₄ (1.9 mg, 0.008 mmol) were mixedin a 1:1 mixture of water and THF (2 ml) was stirred at room temperaturefor 24 h. After evaporation of the solvents, the crude product wasdirectly loaded onto a silica gel column and the product was purified byflash column chromatography, eluting with a 9:1 mixture of CH₂Cl₂ andCH₃OH, to give Compound (XI) as a white solid (12 mg, 75%). HRMSCalculated for [C₁₁₂H₅N₁₃O₅₄S+2Na]²⁺: 1312.4677. Found 1312.4660.Compound (XI) (6 mg, 0.0023 mmol) was dissolved in CH₃OH (1 ml) and asolution of NaOMe in CH₃OH (0.7 M, p. 5 ml) was added. The reactionmixture was stirred at room temperature for 16 h. The reaction wasneutralized by careful addition of Amberlite-15 H⁺ resin and the resinwas filtered. The solvent was removed in vacuo and the residue waspurified by Biogel P-2 gel column chromatography, using water as eluent.The product was lyophilized to give Compound B, where R═H, as a whitesolid (3.7 mg, 87%). HRMS Calculated for [C₇₆H₁₁₉N₁₃O₃₆S+2H]²⁺:911.8873. Found: 911.8820.

EXAMPLE 3 Synthesis of Compound A

A synthesis of Compound A is illustrated in FIG. 6. Compound (XIII) isformed when Compound (XII) (i.e., Acetyl(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)(1-4)(2,3,6-tri-O-acetyl-β-D-galactopyranosyl)(1→4)2,3,6-tri-O-acetyl-α,β-D-glucopyranoside) (96 mg, 0.099 mmol) wasdissolved in 3 ml of dry THF and NH₂NH₂.HOAc (11 mg, 0.119 mmol) wasadded to it. The reaction was stirred at room temperature for 6 h. Thereaction mixture was diluted with 5 ml of EtOAc and 5 ml of water wasadded the organic layer was separated and dried in vacuo to give thehemiacetal (75 mg, 91%), which was directly used in the next step.Anhydrous K₂CO₃ (106 mg, 0.76 mol) was added to the solution ofhemiacetal (70 mg, 0.076 mmol) and trichloroacetonitrile (77 μL, 0.76 mmol) in CH₂Cl₂ (3 ml) at room temperature. The reaction mixture wasstirred at room temperature for 16 h, washed with water, and the organiclayer was dried over anhydrous Na₂SO₄, filtered and concentrated invacuo. The residue was purified by column chromatography, eluting with a1:1 mixture of hexane and EtOAc, to give the trichloroimidate as a paleyellow solid (72 mg, 88%). The imidate (70 mg, 0.065 mmol) and 1-azidohexanol (19 mg, 0.13 mmol) were dissolved in CH₂Cl₂ (2 ml) and cooled to0° C.). A 0.22 M solution of TMSOTf in CH₂Cl₂ (0.13 mmol, 0.2 eq.) wasadded drop wise and the resulting solution was stirred for 1.5 h at 0°C. The reaction was quenched by saturated NaHCO₃ solution (cold) andextracted with CH₂Cl₂. The organic layer was dried over anhydrous sodiumsulfate, filtered, concentrated in vacuo and purified by columnchromatography, eluting with a 3:7 mixture of hexane and EtOAc, to giveCompound (XIII) (i.e.,1-Azido-hexyl(2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl) (1-4)(2,3,6-tri-O-acetyl-β-D-galactopyranosyl) (1→4)2,3,6-tri-O-acetyl-β-D-glucopyranoside) as a solid (38 mg, 68%). HRMSCalculated for [C₄₄H₆₃N₃O₂₆+Na]⁺: 1072.3592. Found: 1072.3586.

Compound (XIII) (19 mg, 0.0183 mmol), a biotin (4 mg, 0.0083 mmol),sodium ascorbate 6 mg, 0.030 mmol), and CuSO₄ (4 mg, 0.014 mmol) weremixed in a 1:1 mixture of water and THF (2 ml) was stirred at roomtemperature for 24 h. After evaporation of the solvents, the crudeproduct was directly loaded onto a silica gel column and the product waspurified by flash column chromatography, eluting with a 8.5:15 mixtureof CH₂Cl₂ and CH₃OH, to give Compound (XIV) as a white solid (14 mg,67%). HRMS Calculated for [C₁₁₂H₁₅₃N₁₁O₅₆S+2H]²⁺: 1290.9664. Found1290.9673. Compound (XIV) (6 mg, 0.0023 mmol) was dissolved in CH₃OH (1ml and a solution of NaOMe in CH₃OH (0.7 M, 0.5 ml) was added. Thereaction mixture was stirred at room temperature, for 16 h. The reactionwas neutralized by careful addition of Amberlite-15 H⁺ resin and theresin was filtered. The solvent was removed in vacuo and the residue waspurified by Biogel P-2 gel column chromatography, using water as eluent.The product was lyophilized to give Compound A, where R═H, as a white,solid (3.4 mg, 84%). HRMS Calculated for [C₇₂H₁₁₃N₁₁O₃₆S+2H]²⁺:870.8608. Found: 870.8644.

As noted herein, synthetic ligands, like the three embodiments ofsynthetic ligands described above, can be used to differentiate betweenvariant Shiga toxins (i.e., Shiga toxin 1 and Shiga toxin 2). Todetermine the selectivity and binding affinities of synthetic ligandsfor any particular variant of the toxin, various detection assay formatsand transducers can be utilized. For example, transducers such as massloading devices (i.e., surface acoustic wave, microcantilevers, surfaceplasmon resonance, interferometric methods), optical devices, andelectrochemical devices can be used. Possible assay formats includesingle binding events as used in mass loading device or sandwich assaysas used in optical sensors or conventional microbiology assays (i.e.,ELISA), luminescence based assay, fluorescence based assay, dipstickassays, or nanoparticles can be used. In one embodiment, an ELISAanalysis was performed on one of the three embodiments of syntheticligands described above. In one particular embodiment, the ELISAprocedure included having the synthetic ligand diluted in either PBS orwater and added to pre-coated microwell plates or containers. In anotherembodiment, these wells can be pre-coated (or treated) withstreptavidin. In this embodiment, the synthetic ligands were thenexposed to an environment having the Shiga toxin for a sufficient periodof time (for example, 2 hours at room temperature). Finally, in thisembodiment, a color was associated with the tested samples against acontrol and analyzed by evaluating the absorbance of the samples usingan ELX800 microplate reader. A general representation showing a toxinattached to the synthetic ligand contained in a well of the assay isshown in FIG. 7. The results of the ELISA procedure are provided inFIGS. 8 a and 8 b, which illustrate the amount of toxin present per wellcontaining a particular synthetic ligand. As shown in FIG. 8 a, theN-acetyl substituted galactosamine for Compounds B and C substantiallybound to the Shiga toxin 2 (serotype (O1117 LPS), while the Shiga toxin1 failed to effectively bind to either compound. In fact, theglycoconjugates associated with Compound B had a greater affinity tobind to Shiga toxin 2 than did those associated with Compound C. Incontrast, FIG. 8 b illustrates that Compound A substantially bound withShiga toxin 1, but did not effectively bind with Shiga toxin 2.

The results shown in FIGS. 8 a and 8 b further support that syntheticligands can be used to differentiate between Shiga toxin 1 and Shigatoxin 2. In fact, the results illustrated in FIGS. 8 a and 8 b indicatethat the N-acetyl groups at the second position for Compounds B and Cprovide the location for the binding with the toxin which issubstantially exclusively with Shiga toxin 2. Moreover, it is importantto note that the embodiments of the synthetic ligands tested anddiscussed above had such high binding-capacities that the resultsindicated the toxins being bound in nanogram quantities from impureculture solutions. Because real world use of such samples and testingprocedures are often times not clean, the binding capacity of theseligands is significant, particularly due to the small quantity of thetoxin which can be detected and identified. Thus, the three embodiedsynthetic ligands discussed above as well as other contemplated andembodied ligands, could be used in hand-held and environmentalbiosensors used in the field. Such synthetic ligands can be used inthese environments because they are stable at ambient temperatures andfor long periods of time. The synthetic ligands provide high levels ofselectivity and sensitivity which can be utilized in diagnostic kits todetect variant forms of the Shiga toxin. One embodiment of such a kitcould include at least one container containing at least once captureagent (i.e., synthetic ligand) which selectively binds to only Shigatoxin 1 or Shiga toxin 2. Such kits could include multiple containerscontaining different capture agents, or an embodiment could include eachkit designated to include a specific capture agent.

Another embodiment for a diagnostic kit is illustrated in FIGS. 9 a and9 b. As shown in this embodiment, the kit 20 includes a base layer 22(i.e., polyolefin flexible film) having a surface 24 which extends froman attached structure 26. The synthetic ligands 28 can be attached tothe surface 24 of the base layer 22. In one embodiment, the surface 24of the base layer 22 is treated with a coating to assist in immobilizingthe synthetic ligand 28. As shown in FIG. 9 a, the synthetic ligand 28(in this example assume Compound B) is exposed to Shiga toxin 2. Thesynthetic ligand 28 binds to the Shiga 2 toxin which increases theamount of material on the base layer 22 which is cantilevered relativeto the attached structure 26, thus causing the base layer 22 to bend.This bending can be quantified and measured providing an indication tothe user that the sample being tested is positive for the Shiga toxin 2.In contrast, as illustrated in FIG. 9 b, using the same embodiedsynthetic ligand 28 (i.e., Compound B) as in FIG. 9 a, but insteadexposing the synthetic ligand 28 to an environment with only Shiga toxin1, finds that the synthetic ligand 28 does not bind to this variant ofthe toxin and thus the base layer 22 does not bend. It is contemplatedthat a variety of methods and systems could be used to measure anddetect the presence or absence of a variant of the Shiga toxin whenusing the synthetic ligands described in claimed herein.

The use of synthetic ligands to detect and quantify the presence orabsence of variants of the Shiga toxin is a significant advancement. Asshown, the examples provided indicate that food products can be analyzedand tested for the variants in the Shiga toxin using synthetic ligands.

EXAMPLE A

Four basic food products were exposed to both Shiga toxin, 1 (Stx1) andShiga toxin 2 (Stx2, including Stx2a). Hamburger and lettuce (1 g each)were suspended in 10 mL of PBS, pH 7.4. The lettuce suspension wassonicated three times for 30 seconds each. The hamburger suspension wasvortexed for approximately 1 min to suspend solids. The milk and applejuice were used undiluted. In previous studies, low pH was found toinfluence glycan binding. The pH of the samples was determined. Thelettuce, hamburger, and milk had a pH of approximately 7.0. Apple juicewas determined to have a pH of 4.0, and was adjusted to pH 7.4 using asmall volume of concentrated sodium hydroxide before use.Glyco-conjugates (for example, Compound A and B described herein) werecoated on strepavidin coated microtiter plates as previously described.Primary rabbit anti-Stx1 and anti-Stx2 (Meridian Biosciences) were usedat a 1:1000 dilution. An ELISA assay was utilized, wherein the secondaryantibody was goat anti-Rabbit IgG labeled with alkaline phosphotase(used at a 1:3000 dilution) and color was detected withp-nitrophenylphosphate using a plate reader at 405 nm. As noted herein,Compound A substantially binds to Shiga toxin 1 and not Shiga toxin 2,while Compound B substantially binds to Shiga toxin 2 and not Shigatoxin 1. Using an ELISA assay, the Shiga toxin 1 was detected in milk,apple, juice lettuce and hamburger, as illustrated in FIG. 10 a.Presence of Shiga toxin 2 was detected in all four food products asillustrated in FIG. 10 b, but detection was reduced in the presence oflettuce and hamburger.

EXAMPLE B

The same food products were tested and prepared as in Example A.However, a luminescent-based assay was used instead where the secondaryantibody was goat anti-Rabbit IgG labeled with horseradish peroxidase(used at a 1:10,000 dilution) and the plate was developed by addition ofluminol reagent mixed with an oxidizer. Here, both Shiga toxin 1 andShiga toxin 2 could be detected in the presence of milk and hamburger,but reduced detection was seen in the presence of apple juice for bothtoxins, and while toxins could be detected in the presence of lettuce, ahigh background signal was observed (see FIGS. 11 a and 11 b).

The foregoing description of embodiments and examples has been presentedfor purposes of illustration and description. It is not intended to beexhaustive or to limit the invention to the forms described. Numerousmodifications are possible in light of the above teachings. Some ofthose modifications have been discussed, and others will be understoodby those skilled in the art. The embodiments as are suited to theparticular use contemplated. It is hereby intended that the scope of theinvention be defined by the claims appended hereto.

What is claimed is:
 1. A method for diagnosing bacterial infectionsmediated by toxins in a patient comprising obtaining a biological samplefrom the patient and assaying for the presence of a toxin in a bindingassay which uses a compound for detecting said toxins, said compoundcomprising the general formula (I):

wherein: n equals 1, 2, 3, 4, 5 or 6, and A comprises a glycoconjugatewherein said glycoconjugate is selected from the group consisting of:

wherein R comprises H; to bind to said toxin; and wherein said toxin isselected from the group consisting of Shiga toxin 1 and Shiga toxin 2.2. A method of detecting a Shiga toxin 1 or a Shiga toxin 2 in food, themethod comprising: exposing a food-based sample to a compound fordetecting said Shiga toxin, said compound comprising the general formula(I):

wherein: n equals 1, 2, 3, 4, 5 or 6, and A comprises a glycoconjugatewherein said glycoconjugate is selected from the group consisting of:

wherein R comprises H; and detecting the presence or absence of saidShiga toxin 1 or said Shiga toxin
 2. 3. The method of claim 2, whereinthe food-based sample comprises milk, apple juice, lettuce or hamburger.4. The method of claim 2, wherein the step of detecting the presence orabsence of Shiga toxin 1 or Shiga toxin 2 is achieved by using anabsorbance based procedure, a luminescence based assay, a fluorescencebased assay, a dipstick assay, or by nanoparticles.
 5. A biologicalassay system for detecting the presence of a Shiga toxin 1 or Shigatoxin 2 toxin comprising: a base layer having a surface; a capture agentcomprising a compound for detecting said Shiga toxin, said compoundcomprising the general formula (I):

wherein: n equals 1, 2, 3, 4, 5 or 6, and A comprises a glycoconjugatewherein said glycoconjugate is selected from the group consisting of:

wherein R comprises H; wherein said compound is characterized by itsability to bind to a receptor site of said toxin, wherein saidglycoconjugate substantially only binds to Shiga toxin 1 or Shiga toxin2, the compound being immobilized onto the surface of the base layer;and a detection system configured to measure when the capture agentbinds with said toxin.
 6. The biological assay system of claim 5,wherein the surface of the base layer has undergone a treatment step toassist in immobilizing the compound.
 7. A method to detect the presenceor absence of Shiga toxin 1 or Shiga toxin 2, the method comprises: a)providing a biological assay system having: i) a base layer having asurface; ii) a capture agent comprising a compound for detecting saidShiga toxin, said compound comprising the general formula (I):

wherein: n equals 1, 2, 3, 4, 5 or 6, and A comprises a glycoconjugatewherein said glycoconjugate is selected from the group consisting of:

wherein R comprises H; wherein said compound is characterized by itsability to bind to a receptor site of the toxin, the compound beingimmobilized onto the surface of the film; and iii) a detection systemconfigured to measure when the capture agent binds with the toxin; b)placing the biological assay system in an environment which may containthe toxin; and c) monitoring the biological assay system for a period oftime sufficient to observe an indication to confirm the presence orabsence of the toxin.
 8. A method for diagnosing bacterial infectionsmediated by toxins in a patient comprising obtaining a biological samplefrom said patient and assaying for the presence of a Shiga toxinselected from the group consisting of Shiga toxin 1 or Shiga toxin 2 ina binding assay which uses a compound for detecting said Shiga toxin,said compound comprising the general formula (I):

wherein: n equals 1, 2, 3, 4, 5 or 6, and A comprises a glycoconjugatewherein said glycoconjugate is selected from the group consisting of:

 wherein R comprises H and said glycoconjugate substantially only bindsShiga toxin 1 and

 wherein R comprises H, thereof and said glycoconjugate substantiallyonly binds Shiga toxin
 2. 9. A method of detecting in food a Shiga toxinselected from the group consisting of Shiga toxin 1 or Shiga toxin 2,the method comprising: exposing a food-based sample to a compound fordetecting said Shiga toxin, said compound comprising the general formula(I):

wherein: n equals 1, 2, 3, 4, 5 or 6, and A comprises a glycoconjugatewherein said glycoconjugate is selected from the group consisting of:

 wherein R comprises H thereof and said glycoconjugate substantiallyonly binds Shiga toxin 1 and

 wherein R comprises H and said glycoconjugate substantially only bindsShiga toxin 2; and detecting the presence or absence of said Shiga toxin1 or said Shiga toxin
 2. 10. A method of detecting the presence in asample of a Shiga toxin selected from the group consisting of Shigatoxin 1 or Shiga toxin 2, said method comprising the steps of: (a)providing said sample; (b) exposing said sample to a first compound fordetecting said Shiga toxin, said compound comprising the general formula(I):

wherein: n equals 1, 2, 3, 4, 5 or 6, and A comprises a glycoconjugatewherein said glycoconjugate is selected from the group consisting of:

 wherein R comprises H and said glycoconjugate substantially only bindsShiga toxin 1 and

 wherein R comprises H and said glycoconjugate substantially only bindsShiga toxin 2; and (c) detecting binding of said first compound.
 11. Themethod of claim 10, further comprising the steps of: exposing saidsample to a second compound for detecting said Shiga toxin, comprisingthe general formula (I):

wherein: n equals 1, 2, 3, 4, 5 or 6, and A comprises a glycoconjugatewherein said glycoconjugate is selected from the group consisting of:

 wherein R comprises H and said glycoconjugate substantially only bindsShiga toxin 1 and

 wherein R comprises H and wherein said glycoconjugate substantiallyonly binds Shiga toxin 2; wherein said second compound substantiallyonly binds a second Shiga toxin; and detecting binding of said secondcompound.
 12. A biological assay system for detecting the presence of aShiga toxin selected from the group consisting of Shiga toxin 1 andShiga toxin 2, said system comprising: a base layer having a surface; acapture agent comprising a compound for detecting said Shiga toxin, saidcompound being immobilized onto said surface of said base layer, saidcompound comprising the general formula (I):

wherein: n equals 1, 2, 3, 4, 5 or 6, and A comprises a glycoconjugatewherein said glycoconjugate is selected from the group consisting of:

 wherein R comprises H and said capture agent substantially only bindsShiga toxin 1 and

 wherein R comprises H and wherein said capture agent substantially onlybinds Shiga toxin 2; and a detection system configured to measurebinding of said capture agent and said toxin.
 13. A method to detect thepresence of a Shiga toxin selected from the group consisting of Shigatoxin 1 and Shiga toxin 2, said method comprising the steps of: (a)providing a biological assay system having: (i) a base layer having asurface: (ii) a capture agent comprising a compound for detecting saidShiga toxin, said compound comprising the general formula (I):

wherein: n equals 1, 2, 3, 4, 5 or 6, and A comprises a glycoconjugatewherein said glycoconjugate is selected from the group consisting of:

 wherein R comprises H and said capture agent substantially only bindsShiga toxin 1 and

 wherein R comprises H and wherein said capture agent substantially onlybinds Shiga toxin 2; and (iii) a detection system configured to measurebinding of said capture agent and said toxin; (b) placing the biologicalassay system in an environment which may contain the toxin; and (c)monitoring the biological assay system for a period of time sufficientto observe an indication to confirm the presence or absence of saidtoxin.